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Site-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) -mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases.

Gene transfer for hemophilia A offers the potential for a one-time disease altering treatment, eliminating the risk of bleeds while freeing patients from the burden of lifelong chronic therapy. SPK-8011 consists of a bioengineered AAV capsid expressing B domain-deleted factor VIII (FVIII) under the control of a liver-specific promoter. In pre-clinical studies, we showed a dose-dependent increase in circulating FVIII levels in non-human primates infused with SPK-8011. We conducted a Phase I/II study of SPK-8011 in 12 men (ages 18-52 years) with severe (n=11) or moderately severe (n=1) hemophilia A. Prior to gene therapy, 8/12 subjects were on prophylaxis, and 4/12 received on-demand treatment. Subjects were enrolled in 1 of 3 dose cohorts, 5E11 vg/kg (n=2), 1E12(n=3), or 2E12(N=7). Safety analysis showed no inhibitor formation. A single serious adverse event (SAE) was reported, associated with an immune response to AAV capsid characterized by simultaneous decline in FVIII, transaminase elevation peaking at Grade 2, and development of positive IFN-g ELISPOTs to capsid was observed beginning at week 6.5 after vector infusion. The asymptomatic transaminase elevation did not respond promptly to initiation of oral steroids and the subject received two infusions of IV methylprednisolone in hospital, thereby fulfilling SAE criteria. The SAE has resolved. All vector doses led to expression of FVIII levels adequate to prevent bleeding and allow cessation of prophylaxis. Across the 12 subjects at 3 doses, there was a 97% reduction in annualized bleeding rate (ABR), and a 97% reduction in annualized infusion rate (AIR). In the 5E11 dose cohort, mean FVIII levels beginning 12 weeks post vector infusion are 13%, with no bleeding events, no elevated transaminase levels, no use of steroids, and stable FVIII expression out to 66 weeks (ongoing). In the 1E12 dose cohort, mean FVIII levels are 15% beginning at 12 weeks post-infusion and stable out to 46 weeks (ongoing). The first subject in the 1E12 dose infused a single dose of factor concentrate for a spontaneous joint bleed at day 159, and the second received multiple infusions for a traumatic bleed beginning at day 195. Declining FVIII levels triggered initiation of a course of tapering steroids in both subjects, at 12 and 7 weeks post vector infusion respectively, which led to stabilization of FVIII levels. The third subject has had no bleeding and did not receive factor infusions or steroids. In the 2E12 (highest) dose cohort, 5/7 subjects currently have FVIII levels 16-49%; their mean FVIII level beginning 12 weeks post-infusion is 30%. No bleeds have been reported among these subjects beginning 4 weeks post vector infusion. Additionally, 5/7 subjects in the 2E12 dose cohort received a course of steroids, initiated at 6-11 weeks post vector infusion, for one or more of the following: declining FVIII levels, rise in ALT above subject baseline, or elevated IFN-g ELISPOTs to AAV capsid. Steroid initiation normalized ALT levels and extinguished the ELISPOT signal in all cases; 2 subjects showed limited stabilization of FVIII levels, which fell to Our data indicate that the kinetics of SPK-8011 expression are similar to those observed with investigational SPK-9001 for hemophilia B. All subjects demonstrated durable transgene expression for up to 66 weeks post vector administration (data cutoff 7/13/18). On cumulative follow up of 345 weeks, SPK-8011 demonstrated a favorable safety profile with no evidence of FVIII inhibitor formation, a single SAE, and 2/12 subjects who experienced ALT elevation above the upper limit of normal that resolved with steroid initiation. Data from the 5E11 (lowest) dose cohort are consistent with published natural history data indicating FVIII:C 12% is adequate to prevent spontaneous bleeding events. Given that 2 subjects in the 2E12 dose cohort lost some FVIII expression, which then stabilized on steroids, and 5/7 subjects in this cohort required steroids, prophylactic steroids may be warranted. We conclude that infusion of SPK-8011 in 12 subjects with severe or moderately severe hemophilia A resulted in safe, durable, dose-dependent FVIII expression resulting in an excellent preliminary efficacy profile with an overall 97% reduction in ABR and AIR. Disclosures High: Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. George:University of Pennsylvania: Equity Ownership; Pfizer: Consultancy. Ragni:CSL Behring: Research Funding; Alnylam: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sangamo: Research Funding; Shire: Research Funding; Biomarin: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Research Funding; Bioverativ: Consultancy, Research Funding; MOGAM: Membership on an entity's Board of Directors or advisory committees; SPARK: Consultancy, Research Funding. Croteau:Novo Nordisk: Consultancy; Octapharma: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Spark Therapeutics: Research Funding; Tremeau Pharmaceuticals: Consultancy; Genetech: Consultancy, Research Funding; CSL-Behring: Consultancy; Catalyst Biosciences: Consultancy; Bioveritiv: Consultancy; Biomarin: Consultancy; Bayer: Consultancy; Baxalta/Shire: Consultancy, Research Funding. Joseney-Antoine:Spark Therapeutics: Employment. Macdougall:Spark Therapeutics: Employment. Tompkins:Spark Therapeutics: Employment. Hait:Spark Therapeutics: Employment. Couto:Spark Therapeutics: Employment. Bassiri:Spark Therapeutics: Employment. Valentino:Spark Therapeutics: Employment. Carr:Spark Therapeutics: Employment. Hui:Spark Therapeutics: Employment. Wachtel:Spark Therapeutics: Employment. Takefman:Spark Therapeutics: Employment. Mingozzi:Spark Therapeutics, Inc.: Employment. Anguela:Spark Therapeutics, Inc.: Employment. Reape:Spark Therapeutics: Employment.

Background: Earlier data demonstrated long-term expression of factor IX (mean FIX:C ~5.1%) following AAV8-mediated gene transfer at 2 x1012 vg/kg in hemophilia B (Nathwani et al., 2014). While the clinical improvement imparted by stable FIX levels is clear, these levels of expression fall short of trough values obtained by long-acting FIX prophylaxis (Santagostino et al. 2016), and of natural history data suggesting that levels of ~12% are required to eliminate spontaneous hemarthroses (den Uijl et al. 2011). Achieving higher levels of FIX:C with dose escalation has not been possible without eliciting a dose-dependent, capsid-specific immune response that may prevent sustained expression and efficacy (Mingozzi et al. 2007, Monahan et al. 2015). We sought to develop a highly efficient vector capsid and expression cassette that could be administered at low doses to achieve hemostatic FIX expression without need for immunosuppression. Methods: The investigational product, SPK-9001, utilizes a bioengineered AAV capsid (Spark100) with liver specific tropism. The prevalence of neutralizing antibodies (NAb) to Spark100 among sampled hemophilia B sera was 40% (Anguela et al. 2015). The expression cassette is a codon-optimized, single-stranded transgene encoding FIX Padua, a naturally occurring variant with a single amino acid substitution (R338L) that confers ~8-fold greater specific activity compared to wild-type FIX (Simioni et al. 2009). Data on bleeding and factor infusions in the year prior to enrollment were retrospectively compiled. Laboratory values, bleeding frequency, FIX consumption, changes in activity and quality-of-life via Haem-A-QoL were prospectively evaluated after vector infusion. Results: We enrolled 9 subjects, of whom 2 failed screening for liver fibrosis and 7 were infused with SPK-9001 at a dose of 5 x1011 vg/kg. Infused subjects were adult males ages 18-52 years with baseline FIX:C 2-34 weeks after vector infusion. Figure 1 outlines subject vector-derived FIX:C for the first 12 weeks. There have been no vector or procedure related adverse events. Steady-state FIX expression is reached by 12 weeks after vector infusion, resulting in a mean FIX:C of 32.3% ±6.5%. To date, no subjects required immunosuppression or demonstrated evidence of a cytotoxic immune response (characterized by loss of FIX activity, elevation of transaminase values >/=1.5-times the upper limit of normal, and positive IFN-gammaELISPOT response to capsid peptides). No subjects developed a FIX inhibitor or demonstrated ELISPOT reactivity to the FIX (R338L) gene product. Subject 3 infused with FIX concentrate for a suspected ankle bleed 2 days after vector infusion. Beyond this, no subjects required factor or experienced any bleeding events. The 4 subjects previously maintained on prophylaxis safely stopped without break-through bleeding. As of today (cumulative 724 days post vector infusion), total factor consumption was reduced by 543,589 IU, tantamount to a cumulative savings of $1,182,298 USD.Six of 7 subjects report increased physical activity and improved quality of life. Conclusion: As of 8/4/2016, we report the highest and most consistent levels of sustained vector-derived FIX:C following FIX gene transfer. Levels of FIX:C achieved by SPK-9001 permitted termination of prophylaxis, prevention of bleeding, and nearly complete cessation of factor use. Despite the heterogeneity in subjects with respect to presence and extent of hemophilic arthropathy, age, and co-morbidities, consistency of transgene expression and clinical outcomes have been observed in all participants studied to date. A vector dose of 5x1011 vg/kg is the lowest dose currently reported in hemophilia gene transfer trials; the absence of any observed CD8+ T cell immune response supports the hypothesis that lowering the dose can reduce or eliminate the risk of a capsid-specific immune response and maximize efficacy. In summary, preliminary data suggest SPK-9001 safely and consistently produces sustained elevation in FIX:C levels sufficient to prevent spontaneous hemarthroses without the need for factor consumption or immunosuppression. Disclosures Ducore: Octapharama: Membership on an entity's Board of Directors or advisory committees; LFB: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Baxalta (Shire): Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees. Cuker:Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding; Genzyme: Consultancy; Stago: Consultancy; Amgen: Consultancy. McGuinn:Spark: Research Funding; Biogen: Research Funding; Novo Nordisk: Research Funding; Baxalta: Research Funding. Luk:Spark Therapeutics, Inc.: Employment. Wright:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: SPK-9001. Chen:Spark Therapeutics, Inc.: Employment. Hui:Spark Therapeutics, Inc.: Employment. Wachtel:Spark Therapeutics, Inc.: Employment. Urich:Spark Therapeutics, Inc.: Employment. Takefman:Spark Therapeutics, Inc.: Employment. Couto:Spark Therapeutics, Inc.: Employment. Carr:Pfizer, Inc.: Research Funding. Anguela:Spark Therapeutics, Inc.: Employment, Patents & Royalties: SPK-9001. High:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: SPK-9001.

Monogenic diseases, including hemophilia, represent ideal targets for genome-editing approaches aimed at correcting a defective gene. Here we report that systemic adeno-associated virus (AAV) vector delivery of zinc finger nucleases (ZFNs) and corrective donor template to the predominantly quiescent livers of adult mice enables production of high levels of human factor IX in a murine model of hemophilia B. Further, we show that off-target cleavage can be substantially reduced while maintaining robust editing by using obligate heterodimeric ZFNs engineered to minimize unwanted cleavage attributable to homodimerization of the ZFNs. These results broaden the therapeutic potential of AAV/ZFN-mediated genome editing in the liver and could expand this strategy to other nonreplicating cell types.

Clinical studies of adeno-associated viral (AAV)-mediated gene transfer of factor IX for hemophilia B have demonstrated long term expression of therapeutic levels of factor IX but revealed that the AAV vector dose may be limiting due to anti-AAV immune responses (Nathwani, 2011). While there is significant interest in moving this approach forward for hemophilia A, it is challenging to express high levels of human factor VIII (hFVIII) due to its intrinsic properties that result in lower expression levels compared to similarly sized proteins (Lynch, 1993). Approaches using codon optimization and variants of hFVIII with enhanced function (increased activity, stability and/or secretion) may provide strategies to increase hFVIII expression to support AAV clinical studies for hemophilia A. For example, we previously developed a codon-optimized hFVIII (CO3) that expressed 5-8-fold higher protein levels than wild type hFVIII after AAV delivery in the context of an optimized expression cassette utilizing a modified transthyretin (TTRm) promoter. Introduction of a PACE-furin (P/F) variant (Siner, 2013) that deletes residues 1645-47 (Δ3) or 1645-48 (Δ4) of the PACE-furin recognition site in CO3 resulted in hFVIII expression after AAV delivery that was 18 (Δ3) or 12-fold (Δ4) better than wild type hFVIII. To date, only one published study has reported clinically relevant levels of human FVIII following AAV treatment in a large animal model. This study used a hFVIII variant that contained a 17 amino acid synthetic sequence flanked by 14-amino acid SQ residues from the N- and C-terminal ends of the B domain (McIntosh, 2013). While the presence of the synthetic spacer allowed for an increase in circulating hFVIII levels, the use of a non-wild-type FVIII sequence in hemophilia A patients may increase the risk of development of neutralizing antibodies to FVIII due to its potential neo-antigenicity. Our goal in this study was to generate an AAV-hFVIII vector capable of expressing therapeutic doses of FVIII at a clinically relevant vector dose without adding any neoantigens to the protein. To this end, we generated 26 codon-optimized hFVIII-SQ constructs under the control of the TTRm promoter. Hydrodynamic delivery of the pAAV-TTRm-hFVIII plasmids identified 11 candidates that expressed FVIII 2-7 fold higher than CO3. Nine of these FVIII expression constructs were made into AAV vectors and delivered to hemophilia A/CD4 KO mice (1x1011 vg/mouse) using a novel capsid, AAV-Spark100. At 4 weeks post vector administration, 2/9 constructs were similar to CO3, 5/9 were 3-4 fold higher than CO3 and 2/9 (SPK-8003 and SPK-8005) were 4-6 fold higher than CO3. To determine if the deletion of the PACE-furin site would result in higher FVIII expression, the Δ4 P/F deletion was introduced into SPK-8003. The levels of FVIII expression after AAV-TTRm-SPK-8003-Δ4 P/F delivery were 2-fold higher than AAV-TTRm-SPK-8003. In order to evaluate the potency of these novel cassettes in a large animal model, SPK-8005 was administered as a single dose via intravenous infusion to male cynomolgus macaques and followed for 8 weeks of observation. At two weeks after gene transfer, NHPs transduced with 2x1012 vg/kg of SPK-8005 expressed hFVIII antigen levels of 12.7 ± 2.1% (average ± standard error of the mean, n=3). Average FVIII expression after treatment with 5x1012 vg/kg was 22.6 ± 0.8% (n=2). Finally, at the highest tested dose of 1x1013 vg/kg, hFVIII antigen levels of 54.1 ± 15.6% were observed two weeks after AAV infusion (n=3). As anticipated, hFVIII expression declined in approximately one third of the animals around week 4, concomitant with the appearance of inhibitory antibodies to human factor VIII in these macaques. In summary, these data using highly active, novel codon-optimized FVIII constructs devoid of potential neoantigens demonstrate the feasibility of lowering the AAV capsid load for a gene-based therapeutic approach for hemophilia A to a dosage level that appears to be efficacious and safe in the treatment of hemophilia B. Disclosures Anguela: Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Elkouby:Spark Therapeutics, Inc.: Employment, Equity Ownership. Toso:Spark Therapeutics, Inc.: Employment, Equity Ownership. DiPietro:Spark Therapeutics, Inc.: Employment, Equity Ownership. Davidson:Spark Therapeutics: Consultancy. High:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: AAV gene transfer technology. Sabatino:Spark Therapeutics, Inc.: Research Funding.

Genome editing has the potential to provide long-term therapeutic gene expression in vivo. We have previously demonstrated efficient editing in a mouse model of hemophilia B through liver-directed adeno-associated viral vector (AAV) delivery of a zinc finger nuclease (ZFN) pair and a corrective donor. We determined that homology is not necessary to achieve efficient levels of genome editing in adult mice, consistent with the fact that quiescent cells, including adult hepatocytes, are not thought to be amenable to homology directed repair (HDR). As a consequence of the donor containing a splice acceptor, both HDR and homology independent vector integration are capable of driving human factor 9 (hF.IX) expression. In this study we sought to determine whether hF.IX expression in mice treated as neonates, undergoing substantial hepatocyte proliferation, is predominantly the result of HDR or homology independent genome editing. Provided the efficacy is not substantially reduced, an HDR dependent approach would impose additional constraints on targeting. Treatment of neonatal hF9mut mice (harboring the ZFN target site) with 1x1011 vg AAV8-ZFN and 5x1011 vg AAV8-Donor via retro-orbital injection resulted in a drastic difference in hF.IX expression between donors with and without homology 10 weeks post injection (Homology: 1531 ± 174.5 ng/mL vs. No-homology: 146.1 ± 5.8 ng/mL; n=12 and 7, respectively). We next asked whether HDR could be stimulated even more specifically through the induction of DNA single strand breaks at the target site. We treated neonatal mice with homologous or non-homologous donors, as well as ZFNs or ZFNickases (in which one FokI nuclease domain was inactivated with the D450A mutation). ZFNickases were indeed active, resulting in ~250 ng/mL hF.IX 4 weeks post injection (Figure 1). Interestingly, we could not detect hF.IX in mice treated with ZFNickase and no-homology donor (LOD: 15ng/mL). To rule out the possibility that this was simply due to the lower efficacy of ZFNickases compared to ZFNs, we increased the ZFNickase dose 4 fold. Four weeks post treatment, we observed substantial levels of hF.IX in mice treated with homologous donor (2041 ± 269 ng/mL) and were again unable to detect hF.IX in mice treated with the non-homologous donor (n=10 and 7, respectively). These data point to homology directed repair as the primary mechanism of protein production for genome editing in neonatal mouse liver, and suggest improvements in both efficacy and specificity can be made through deeper understanding of the molecular requirements of this approach. Figure 1. Figure 1. Disclosures Anguela: Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Doyon:Sangamo BioSciences: Employment. Wechsler:Sangamo BioSciences: Employment. Paschon:Sangamo BioSciences: Employment. Davidson:Spark Therapeutics: Consultancy. Gregory:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. High:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Genome editing utilizing engineered zinc finger nucleases (ZFNs) is a promising approach to achieve long-term expression of therapeutic genes in vivo. We have previously demonstrated in vivotargeting of the endogenous murine albumin locus as a “safe harbor” for high levels of protein production, resulting in sufficient Factor IX to correct the disease phenotype in hemophilia B mice. Targeted insertion of the donor sequence into the genome offers multiple advantages. First, we are able to exploit the high transcriptional activity of the native albumin enhancer/promoter. Second, by obviating the need for these regulatory elements within the donor, we expand the effective carrying capacity of adeno-associated viral (AAV) vectors to enable delivery of larger transgenes that may not package efficiently, such as coagulation factor 8. B-Domain Deleted Factor VIII (BDD-F8) cDNA is approximately 4.4kb. Inclusion of required enhancer/promoter elements results in a construct that exceeds the ideal packaging limitations of rAAV vectors. However, since these regions are not required for our gene editing approach the promoterless hBDD-F8 donor remained below the AAV packaging capacity. Importantly, intravenous delivery of 5e11 vg of AAV8-mAlb-ZFN and 5e11 vg of AAV8-BDD-F8-Donor to hemophilia A mice resulted in 54.6% (±4.1%) FVIII activity in the blood 2 weeks following administration - confirming the potential of the albumin locus to express high levels of the targeted transgene. To further optimize in vivo ZFN-based genome editing with a view toward the ultimate clinical use of this technology, we sought methods to further increase ZFN potency (and thus limit the dose of AAV necessary for function). Of several strategies pursued (e.g. codon optimization and inclusion of a intron in the expression cassette) the most successful was to deploy separate vectors expressing each individual ZFN rather than a single vector encoding a dual expression cassette carrying both ZFNs separated by a 2A fusion peptide. Using next generation sequencing (Illumina’s MiSeq) technology to quantify insertions and deletions indicative of DNA cleavage and repair, we observed a >3-fold increase in ZFN potency in vivo by transitioning from the dual expression vector to two individual ZFN vectors at equivalent total vector doses. Given the encouraging results obtained in mice, we next sought to examine the effectiveness of targeting the albumin locus in non-human primates (NHPs). Importantly, a single intravenous co-injection of two individual AAV vectors encoding each of the NHP targeted albumin-specific ZFNs resulted in persistent levels of gene modification in liver biopsies from treated Rhesus macaques - demonstrating successful in vivocleavage in a large animal model. These data support the use of ZFN technology in the targeting of endogenous loci with large therapeutic transgenes that are not ideally suited for episomal AAV based expression (such as F.VIII). Together our results support the further investigation of genome editing at the albumin locus as a novel method for in vivo protein replacement. Disclosures: Doyon: Sangamo BioSciences, Inc.: Employment. Wechsler:Sangamo BioSciences, Inc.: Employment. Paschon:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment. High:Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Intrexon: Consultancy; Genzyme, Inc.: Membership on an entity’s Board of Directors or advisory committees; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; BioMarin: Consultancy; Alnylam Pharmaceuticals: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Shire : Consultancy; Benitec: Consultancy.

Abstract 751 Gene correction using zinc finger nuclease (ZFN) technology can be applied to target virtually any locus in the human genome. Beyond correcting mutated genes causative of disease, ZFNs can also be utilized to target transgene insertion into genomic “safe harbors.” Ideally, specific gene targeting to such “safe harbor” sites would (i) ensure therapeutically relevant levels of transgene expression and (ii) tolerate transgene addition without deleterious effect on the host organism. For liver-derived protein replacement, albumin represents an attractive target locus. Firstly, albumin is very highly expressed exclusively in the liver, thus targeting of a relatively small percentage of alleles should yield therapeutically relevant levels of liver-specific transgene expression. Second, the reduction or complete absence of albumin in animals and even humans (analbuminemia) produces surprisingly few symptoms. Here, we sought to investigate whether ZFN-mediated targeted insertion of a promoter-less copy of the human F9 cDNA at the mouse albumin locus could result in human Factor IX production and successfully correct the hemophilic phenotype in mice. To address this question, we constructed an AAV vector encoding a pair of ZFNs targeting intron 1 of the mouse albumin locus (AAV8-mAlb-ZFN) and a donor AAV vector (AAV8-Donor) harboring a partial cDNA cassette containing exons 2–8 of the wild-type human F9 gene flanked by sequences lacking significant homology to the mouse genome. Co-delivery of 1e11 vg of AAV8-mAlb-ZFN along with 5e11vg of AAV8-Donor resulted in stable (>12wk) circulating F.IX levels of 1600–3200 ng/mL (32–64% of normal). As a control, mice injected with the AAV8-Donor along with an AAV vector encoding a ZFN pair targeting an unrelated locus exhibited background F.IX levels (∼50 ng/mL). A dose-response study was performed by administering a fixed dose of donor (5e11 vg/mouse) with decreasing doses of AAV8-mAlb-ZFN (1e11, 1e10 and 1e9 vg/mouse). Human F.IX levels increased as a function of ZFN dose in the range tested (3260±480, 225±43 and 31±4 ng/mL at the high, medium and low dose, respectively). Importantly, these results showed that donor homology to the target site is not required to achieve robust levels of gene addition to the albumin locus in adult mice, thus permitting the design of donor vectors harboring corrective copies of transgenes up to the maximum AAV packaging capacity of ∼4.7 Kb. Albumin and factor IX are both synthesized as pre-propeptides and turned into propeptides after the signal peptide is removed. Expression of human F9 exons 2–8 spliced with mouse albumin exon 1 is expected to yield a chimeric propeptide. The first 2 N-terminal amino acids would originate from proalbumin, followed by a Val to Leu mutation at position −17 of the hF.IX propeptide and 16 aa encoded by human F9. To evaluate whether this chimeric human F.IX derived from gene addition to the albumin locus would be processed correctly and normalize the prolonged clotting times in hemophilia B (HB) mice, we injected 1e11 vg of AAV8-mAlb-ZFN and 5e11vg of AAV8-Donor into HB animals. Two weeks post-treatment, hF.IX antigen levels were in the range of 20% of normal and activated partial thromboplastin time, a measurement of clot formation, was corrected to wild-type levels (42 seconds), from an average of 70 seconds pre-treatment. Thus expression of a therapeutic protein (F.IX) from the albumin locus is shown to correct the HB disease phenotype in vivo. In summary, these data provide the first demonstration of ZFN-mediated in vivo genome editing of a safe harbor locus for therapeutic protein production. While we provide here a proof of principle establishing phenotypic correction of hemophilia B, appropriately designed donors could expand this strategy. Most importantly the magnitude of albumin expression (>15 g / day) should enable production of a diverse range of transgenes at therapeutically consequential levels. Disclosures: Anguela: The Children's Hospital of Philadelphia: Patents & Royalties. Sharma:The Children's Hospital of Philadelphia: Patents & Royalties. Doyon:Sangamo BioSciences, Inc.: Employment. Wong:Sangamo BioSciences, Inc.: Employment. Paschon:Sangamo BioSciences, Inc.: Employment. Gregory:Sangamo BioSciences, Inc.: Employment. Holmes:Sangamo BioSciences, Inc.: Employment. Rebar:Sangamo BioSciences, Inc.: Employment. High:Shire Pharmaceuticals: Consultancy; Sangamo Biosciences, Inc: Collaborator, Collaborator Other; Novo Nordisk: Visiting Professor, Visiting Professor Other; Genzyme, Inc: Membership on an entity's Board of Directors or advisory committees; The Children's Hospital of Philadelphia: Patents & Royalties; Bluebird Bio, Inc: Membership on an entity's Board of Directors or advisory committees.

Abstract 2050 Adeno-associated viral (AAV) vector-mediated gene transfer has shown great potential as a therapeutic platform for inherited and metabolic diseases. Systemic delivery of AAV vectors through the bloodstream is a safe, non-invasive, and potentially effective strategy to target a variety of organs, including liver, muscle, and brain. However, neutralizing antibodies (NAb) to AAV, highly prevalent in humans, constitute a major obstacle to successful gene transfer, particularly when a vector is delivered through the vasculature. Thus far, the liver was targeted to express the coagulation factor IX (F.IX) transgene in two clinical studies. In one study, a single-stranded AAV2 vector expressing the F.IX transgene was delivered through the hepatic artery to severe hemophilia B subjects at doses of 8×1010, 4×1011, and 2×1012 vector genomes (vg)/kg. Efficacy was observed in one subject from the high-dose cohort, who achieved peak F.IX transgene plasma levels of ∼10% of normal. The subjects infused at lower doses did not show any evidence of transgene expression, despite the fact that they did not have detectable NAb to AAV. In a second study, a self-complementary AAV8 vector expressing the F.IX transgene was delivered through peripheral vein infusion to severe hemophilia B subjects at doses similar to those administered in the AAV2 study, 2×1011, 6×1011, and 2×1012 vg/kg. All subjects enrolled in the AAV8 trial had evidence of transgene expression above baseline levels, despite the fact that some of the subjects had low-but-detectable anti-AAV8 NAb. Peak F.IX plasma levels at the high vector dose were 8–12% of normal, similar to the high dose of the AAV2 trial, suggesting that the vectors used in the two studies had comparable potency. Importantly, the vectors used in the two studies differed in empty capsid content, as the AAV2 vector preparation was essentially empty capsid-free and the AAV8 vector contained a 5–10 fold excess of empty capsids. The current study was undertaken to explore the role of empty capsids as a factor in the difference in outcome in the low- and mid- dose cohorts of the two trials. Our underlying hypothesis was that the presence of an excess of empty capsids effectively absorbs low-level neutralizing and non-neutralizing antibodies, and permits transduction even in their presence. Using a newly developed AAV antibody dot-blot assay, we demonstrate that adult human subjects with a low to undetectable NAb titer (1:1) as assessed by a commonly used assay do, in fact, carry significant amounts of anti-AAV antibodies. Conversely, children aged one year appear to be truly naïve for anti-AAV humoral immunity. Using C57BL/6 mice passively immunized with purified human IgG injected intraperitoneally 24 hours before vector administration, we further demonstrate that the same low levels of anti-AAV antibodies found in humans (NAb titer of 1:1–1:3) can block >90% of liver transduction after peripheral vein delivery of AAV8 vectors expressing F.IX at doses of 1×1012 vg/kg, comparable to those tested in the clinic. We next demonstrated that the inhibitory effect of low titer (1:1–1:3) anti-AAV antibodies can be overcome by adding a 5 to 10-fold excess of empty capsids to the final formulation of AAV8 vector, and that empty capsid content can be carefully titrated as a function of the animal's anti-AAV NAb in order to achieve efficient target organ transduction, even at titers >1:100. However, the beneficial effect of empty capsids on liver transduction is lost when a 1000-fold excess of AAV8 empty capsids are added to the formulation of AAV8 vectors, due to receptor binding competition. This inhibitory effect could be avoided by using AAV2 empty capsids, which efficiently protect AAV8 vectors from NAb without inhibiting transduction. These results were confirmed in non-human primates, a natural host for AAV8, in which a 5 to 6-fold increase in liver transduction was achieved by formulating vector in 5–10 fold excess AAV8 empty capsids, reaching levels of F.IX expression of 10 to 20% of normal. Application of these findings to the development of personalized formulations of vector product for intravascular delivery will facilitate safe, effective AAV-mediated gene transfer in settings in which vectors are delivered through the systemic circulation. Disclosures: Mingozzi: Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. Anguela:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. Wright:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. High:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties.

Abstract 668 As a therapeutic strategy, site-specific modification of the genome has the potential to avoid some of the disadvantages of traditional gene replacement approaches such as insertional mutagenesis and lack of endogenous regulatory control of expression. We have recently reported that zinc finger nuclease (ZFN) driven gene correction can be achieved in vivo in a neonatal mouse model of hemophilia by combining AAV-mediated delivery of both the ZFNs and a Factor IX donor template with homology to the targeted F.IX gene (Li et al., Nature, 2011). The mouse model carries a mutant human F.IX mini-gene (hF9mut) knocked into the ROSA26 locus and ZFN-mediated cleavage followed by donor-dependent repair results in restoration of functional F.IX expression. AAV-ZFN and AAV-Donor vectors were administered to neonatal mice, where the rapid proliferation of hepatocytes in the growing animal may promote genome editing through homology directed repair (HDR). Here we sought to investigate whether ZFN-mediated genome editing is feasible in adult animals with predominantly quiescent hepatocytes. Tail vein injection of the AAV-ZFN and AAV-Donor, containing a promoterless wild type factor IX insert flanked by arms of homology to the target site, into adult (8 week old) mice (n=17) resulted in stable (>10wk) circulating F.IX levels of 730–1900 ng/mL (15-38% of normal), whereas mice receiving ZFN alone (n=9) exhibited F.IX levels below detection ( In summary, we have shown that synchronized cell proliferation of hepatocytes, either in neonatal mice or following partial hepatectomy, is not necessary to achieve highly efficient genome editing and resultant high levels of transgene expression in vivo. These findings substantially expand the potential of ZFN-mediated genome editing as a therapeutic modality. Disclosures: Doyon: Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment.

Abstract LBA-5 Inherited hematologic disorders have the potential to be effectively treated by gene therapy, with recent successes reported for several genetic disorders using viral vector-mediated gene transfer (ADA-SCID, NEJM 2009; β-thalassemia, Nature 2010). However, these trials and others illustrate some of the disadvantages and risks of using viral vector-based gene addition strategies, including loss of endogenous gene regulation and random insertion leading to potential for insertional mutagenesis. An alternative approach is gene correction, where in situ correction of a gene mutation allows endogenous gene regulation and decreases risks related to random integration. Gene correction is based on gene targeting, the therapeutic utility of which has historically been limited to mouse embryonic stem cells due to low homologous recombination rates in other cell types. However, a recently developed class of fusion proteins, zinc finger nucleases (ZFNs), have been shown to increase targeting efficiency 2–3 logs by inducing site-specific DNA double strand breaks at the intended targeting site. ZFNs have permitted high efficiency therapeutic gene targeting in a variety of cultured cells previously thought intractable to these processes, but ZFN-mediated gene correction has yet to be successfully achieved in vivo in an animal model of disease. Here we show ZFN-mediated therapeutic gene targeting of a mutated F9 gene in vivo, resulting in phenotypic correction of a mouse model of hemophilia B (HB). We first generated ZFNs targeting intron 1 of the human F9 gene (F9 ZFNs). We hypothesized the F9 ZFNs would mediate insertion of a wild-type F9 exons 2–8 minigene into intron 1 via gene targeting, thus bypassing the 95% of F9 mutations that occur in exons 2–8. We next generated a humanized HB mouse model with a deletion of the mouse F9 gene and knock-in (at the ROSA 26 locus) of a catalytic domain-deleted human F9 mini-gene (hF9mut) transgene. Adeno-associated viral (AAV) vector delivery of the F9 ZFNs to hF9mut mouse liver resulted in cleavage of the intron 1 target site in 45% of hepatocytes. We then generated an AAV donor vector containing a w.t. exons 2–8 insert flanked by arms of homology. Co-delivery of the AAV-ZFN and AAV-donor vectors to neonatal hF9mut mice (n=16) resulted in circulating F.IX levels of 120–350 ng/mL (2-7% of normal), whereas mice receiving AAV-ZFN alone (n=17) or AAV-mock & AAV-donor (n=15) had no detectable F.IX expression (detection limit 15 ng/mL), or Disclosures: Doyon: Sangamo Biosciences: Employment. Li:Sangamo Biosciences: Employment. Wong:Sangamo Biosciences: Employment. Paschon:Sangamo Biosciences: Employment. Rebar:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo: Employment. High:Sangamo Biosciences: Consultancy; Children's Hospital of Philadelphia: Patents & Royalties.